Resazurin metabolic assay

Resazurin metabolic assays are used to measure the metabolic rate of marine organisms. This protocol has been developed for use in oysters, but can be adapted for other organisms.

Contact: ashuff (at) uw (dot) edu

Standard Operating Protocol (SOP)

Written 20250401 by Ariana Huffmyer.

Overview

This protocol is written for small oyster spat, but can be adapted for other life stages. This protocol is based on previous work in the lab by Colby E. and as used in our analysis of oyster seed metabolic response and survival.

Materials and preparing solutions

  1. Stock resazurin solution

To make the resazurin stock solution (10 mL) mix the following. We will use this solution for multiple trials.

Store in a dark fridge or freezer.

  1. Working resazurin solution

First, determine the volume of resazurin required depending on the size of your organism and the container size. Here are some examples that we have used before. We recommend performing preliminary trials to ensure that a change in resazurin fluorescence can be detected over the time scale desired. This protocol was developed to detect a change in resazurin fluorescence within 5 hours in small seed (<7mm) and larger seed (10-30mm).

  • Small seed (<7mm length): trials conducted in 96 well plates (300uL volume in each well)
  • Medium seed (15-40 mm length): trials conducted in small plastic cups (20 mL volume)
  • Large seed/adults (>40 mm length): trials can be conducted in tripour cups, beakers, or plastic cups. Scale volume appropriately. The animals should be fully submerged.

To prepare the working solution of resazurin, prepare the following. 

  • Desired working volume x 0.98666 = ___ mL filtered seawater (DI water with Instant Ocean adjusted to 23-25 ppt or filtered (<1um) seawater) 

  • Desired working volume x 0.00222 = ___ mL resazurin stock solution as made above in step 1 

  • Desired working volume x 0.001 = ___ mL DMSO

  • Desired working volume x 0.01 = ___ mL antibiotic solution 100x Penn/Strep & 100x Fungizone - this should be kept frozen in a dark freezer and thawed before use (thaw in the dark or cover with aluminum foil)

To prepare the working solution of resazurin, prepare the following.

This recipe is to make 150 mL of working stock. To run a single 96 well plate, approximately 35 mL will be required (allowing for extra for re-do’s or errors). For example, if we are running 4 total plates, we will need to prepare 4 x 35 mL = 150 mL of working solution. Increase if more is required.

  • 148 mL seawater (DI water with Instant Ocean adjusted to 23-25 ppt or filtered (<1um) seawater)
  • 333 µL resazurin stock solution as made above in step 1
  • 150 µL DMSO
  • 1.5 mL antibiotic solution 100x Penn/Strep & 100x Fungizone

Store at 4°C in dark fridge until use. We recommend making a fresh batch of working stock within 7 days of use.

  1. Supplies
  • Bench top incubators
  • Temperature loggers to be placed in incubators at treatment conditions
  • Paper towels and bench paper/pads
  • Tweezers, transfer pipettes, and forceps
  • Dissecting microscope
  • Spectrophotometer plate reader that detects fluorescence (e.g., FLx800) and software
  • Plate reader filters with excitation wavelength of 530 and emission wavelength of 590 (we are currently using an excitation 528 wavelength filter with a bandpass of 20 and an emission 590 wavelength filter with a bandpass of 20)
  • P1000 pipette
  • Scale bar/ruler
  • Camera/phone camera
  • Plastic cups, beakers, plates, or other vessel for incubations
  • 96 well plate (for taking readings, you will need this regardless of container type)

Label plates with identifying number (e.g. “Plate 1”, “Plate 2”) or label cups with unique numbers/identifiers.

Protocol

Conduct measurements at treatments desired. We typically conduct measurements at a control temperature and a high temperature each day. If multiple treatments are desired over multiple days, be sure to run a control treatment each day as reference. Ensure you account for tank effects or other batch effects by randomizing loading order, position in incubators, etc.

For oysters, we recommend the following treatments:

  • For acute stress and survival testing: control temperature (10-20°C) and acute high temperature (40-44°C)
  • For thermal performance testing: control temperature (10-20°C), 36°C, 38°C, 40°C, 42°C

Schedule

Each day, the schedule will be as follows. Note that this schedule is written for a single person. If there are 2-3 people, the time frame for loading and assessing survival will be shorter than written here.

08:00-09:00: Load plates with oysters, take size images, and load resazurin solution
09:00: Time 0 measurement
10:00: Time 1 measurement
11:00: Time 2 measurement
12:00: Time 3 measurement
13:00: Time 4 measurement
14:00: Time 5 measurement 14:00-16:00: Survival assessments and clean up

Note that it is critical to perform survival assessments so that you can analyze resazurin metabolic response for those that survive and those that die during the trials. If performing trials in 96 well plates or other small containers, we recommend performing survival assessment at the end of the incubation. If you are conducting trials in larger cups where you can see the animals without removing the resazurin solution, you can assess survival at each time point.

Load and prepare samples

Before starting, set the incubator at the desired temperature.

  1. Prepare animals for assays. Track the source tank, treatment, or other identifying information.
  2. Add animals into labeled plates or containers and placing them into the empty container.
  3. Have at least n=6 empty wells/containers at each temperature to serve as blanks.
  4. Write the location of wells on a plate map if using plates.
  5. Allocate the animals either into their designated cup or onto the lid of the plate.
  6. Take images of each animal with a scale bar with their identifying information in the photograph. See an example below.
  7. Move the animals into their respective wells in the plate if you placed them on the lid for a photograph.
  8. Fill each well with the desired amount of resazurin working stock at ambient temperature using a microchannel pipette or graduated cylinder.

An example of photograph for size measurements:

Measurements

  1. Turn on the computer and plate reader. Open the plate reader software.
  2. Create a new protocol that conducts end point measurements from the top of the plate using an excitation wavelength of 530 and emission wavelength of 590 nm.
  3. Name the protocol and save. This process may vary depending on your instrument.
  4. Take a T0 initial measurement - this is critical! If using a 96-well plate with small animals, you can place the plate with the animals directly on the plate reader (do not have the lid on the plate). If you have animals in cups, take a small sample (250uL) of the resazurin liquid from each container, place into a 96-well plate, and then conduct the measurements. If you use this method, be sure to make a plate map of the location of the samples and identifying information. Conduct measurements for blanks and samples.
  5. Put the first plate on the loading platform.
  6. Collect and export readings as directed in the plate software.
  7. Save the file as: YYYYMMDD_TemperatureTreatment_Plate#_T0.xlsx. For example, 20250128_40C_Plate#_T3.xlsx.
  8. Save the data to a flash drive and add to GitHub or data repository. Here is an example of our files at the GitHub repository here.
  9. Record the time of the measurement.
  10. Repeat for any remaining plates or treatments.
  11. Move the animals to the incubator at their respective temperatures and record the temperature in the incubators.
  12. Repeat at 1, 2, 3, 4, and 5 hours of incubation. A minimum incubation time is 4 hours with 5-6 hours as optional time points.

Here is an example of the plate maps from our work.

Survival measurements

  1. Either each hour (if you can easily see the animals in larger cups) or at the end of the incubation (for animals in 96 well plates), conduct survival assessments.
  2. If using plates, prepare a plate map for recording the assessments - this is an easy method to keep track of the assessments. If you are not using plates, make a list of all samples with columns for recording survival at each time point.
  3. In the plate maps, you will record which wells have dead oysters. Use an “X” to mark wells with dead oysters. You can also record “dead” and “alive” in a list format.
  4. Start with high temperature plates.
  5. If working with shellfish, use tweezers/forceps to take each animal out and examine in a petri dish filled with DI water under a dissecting scope. Determine if the oyster is dead by placing the cup side of the oyster down and gently taping/moving the shell. If the shell is open and remains open after tapping, the oyster is dead. If the shell is closed tight or closes after tapping, the oyster is alive. Use other determination methods for other organisms.
  6. Record any notes of oysters that were damaged by the tweezers or record any other notes of interest.
  7. After examining each oyster, move it to a beaker. Discard oysters after the measurements are done.
  8. Repeat for all plates and samples.
  9. Discard of resazurin in the appropriate waste bin labeled for hazardous waste. Empty plates into a tripour and pour into a labeled waste container.
  10. Rise plates thoroughly in the sink and allow to dry for the next trial. Cups and materials can be re used.
  11. Generate a data frame that has columns for sample ID, treatments, date, and other relevant information. Add a column designated “mortality” and add a 0 for alive and 1 for dead animals. See examples below.

Here is an example of the data sheet in the notebook from our work.

Size measurements

From the images, measure the size of the organism. For oysters, we often use maximum length (mm). Other measurements may be more appropriate for other organisms. This will be used to normalize resazurin measurements.

Record size from measurements of images (e.g., using ImageJ) in a data frame. See examples below.

Data preparation and analysis

Prepare the following data frames (see examples at the links below):

  • Size measurements: columns for sample ID, date, treatment, and size measurement (e.g., length in mm)
  • Mortality assessment: columns for sample ID, date, treatment, and mortality assessment (e.g., 0 for alive and 1 for dead)
  • Metadata: columns for sample ID, date, treatment, tank or batch effects, species, well/cup ID, and sample type (i.e., “blank” or “sample”)
  • Resazurin files: files exported from plate reader software that contain fluorescence readings for each well of the plate

Conduct the following analysis steps (see R scripts available for use below):

  • Read in data
  • Normalize all fluorescence values to the initial time point (fluorescence at time X divided by fluorescence at time 0)
  • Calculate the mean value for blanks within each batch (e.g., mean of all blanks in plate 1 at high temperature on day 1)
  • Subtract the mean blank value from the fluorescence value of each sample from the respective batch
  • Size normalize the data by dividing fluorescence values by size of each sample
  • Proceed with visualization and statistical analyses, including testing for effects of treatment or other effects of interest and examining metabolic differences between animals that lived and animals that died during the trials. See examples in our GitHub repositories linked below.

Data sheet and script examples

Data will be stored on GitHub. Links are available below for examples.

Size image examples
Size data sheet example
Resazurin plate reader file examples
Resazurin plate metadata example
Survival data example

Small seed project example: Scripts for analysis are available on GitHub here and figures of results are available here.

Large seed project example: Scripts for analysis are available on GitHub here and figures of results are available here.

Aquarium nitrogen testing kit protocols (AQI test kit)

Written by Noah Ozguner and Madeliene Baird 20250401.

This protocol is written based on the manufacturer’s instructions.

Test kit was ordered here

Standard Operating Protocol (SOP)

After testing, do not pour contents back into the tank. Make sure to rinse test tubes after use.

pH (kit minimum 7.4, maximum 8.8)

  • Fill a clean test tube with 5 ml of water
  • Add 5 drops of High range pH Test Solution
  • Cap the test tube and invert it multiple times to mix the solution
  • Read results by comparing the color of the solution to the provided pH color chart (in a relatively well lit area). The closest color match indicates the pH value of the water sample.
  • Thoroughly wash test tube after use

Ammonia (ppm or mg/L) [two bottles]

  • Fill a clean test tube with 5 ml of water
  • Add 8 drops of Ammonia Test Solution 1
  • Add 8 drops of Ammonia Test Solution 2
  • Cap the test tube and shake vigorously for 5 seconds
  • Wait 5 minutes for color to develop
  • Read results by comparing the color of the solution to the provided Ammonia color chart. (in a relatively well lit area). The closest color match indicates the Ammonia value of the water sample.
  • Thoroughly wash test tube after use

Nitrite (ppm or mg/L)

  • Fill a clean test tube with 5mL of water
  • Add 5 drops of Nitrite Test Solution
  • Cap the test tube and shake for 5 seconds
  • Wait 5 minutes for color to develop
  • Compare the color of the solution to the Nitrite Color Chart in a well lit area. The closest match will indicate the ppm (mg/L) of the nitrite in the sample
  • Thoroughly wash test tube after use

Nitrate (ppm or mg/L) [two bottles]

  • Fill a clean test tube with 5mL of water
  • Add 10 drops of Nitrate Test Solution #1
  • Cap the test tube and invert several times to mix the solution
  • Vigorously shake the Nitrate Test Solution #2 for at least 30 seconds
  • Add 10 drops of Nitrate Test Solution #2
  • Cap the test tube and shake vigorously for 1 minute
  • Wait 5 minutes for the color to develop
  • Compare the color of the solution to the Nitrate Color Chart in a well lit area. The closest match will indicate the ppm (mg/L) of the nitrite in the sample
  • Thoroughly wash test tube after use

Reference values

Ammonia (NH3): Ammonia is highly toxic to fish and should always be 0 ppm.

Nitrite (NO2): Nitrite is also toxic and should be 0 ppm.

Nitrate (NO3): While less toxic than ammonia and nitrite, nitrate can still be harmful to fish and corals at high levels. Aim to keep nitrate levels below 20 ppm, and ideally between 0-5 ppm.